Pak. J. Pharm. Sci., Vol.29, No.5, September 2016, pp.1519-1523 1519 Quantification of polyphenolic compounds and flavonoids in Achillea millefolium and Equisetum arvense Muhammad Nasimullah Qureshi 1* , Guenther Stecher 2,3 and Guenther Karl Bonn 2 1 Department of Chemistry, Abdul Wali Khan University Mardan, Mardan, Pakistan 2 Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University Innsbruck, Innrain Innsbruck, Austria 3 Bionorica Research GmbH, Mitterweg, Innsbruck, Austria Abstract: Flavonoids have been of considerable importance and interest because of their medicinal activity. Responding to their numerous health benefits, a comparative study on the quantitative determination of total polyphenolic compounds and flavonoids was carried out in Achillea millefolium and Equisetum arvense. Total polyphenolic compounds were quantified by Folin-Ciocalteau method using different solvents in order to prove their extraction efficiency. Focus within total polyphenolic quantification study was placed on the traditional reflux and solvents used were: water, 100% acetone, 100% ethanol, 80% ethanol, 50% methanol and 70% methanol. In order to make flavonoids free from glycosidic moiety for quantification, hydrolysis was performed in 50% MeOH at 90°C using 6 M HCl concentration. Reverse phase high performance liquid chromatography (RP-HPLC) in gradient elution mode at 50°C using Hypersil BDS (RP-18) column was employed for the separation of flavonoids. Mobile phase used consisted of different combinations of water-methanol-tetrahydrofuran-phosphoric acid. Flavonoids quantified were luteolin, quercetin, apigenin, isorhamnetin and kaempferol. Keywords: Medicinal plants, Polyphenolic compounds, Flavonoids, Hydrolysis, HPLC-PDA. INTRODUCTION Medicinal herbs and herbal products have been of considerable vitality for their various biologically and pharmacologically important chemicals (Qureshi et al., 2012a; Qureshi et al., 2011a; Qureshi et al., 2013; Qureshi et al., 2014a; Qureshi et al., 2014b; Qureshi et al., 2012b; Qureshi et al., 2011b). Flavonoids are products of secondary metabolism and are polyphenolic in their chemical structure. They are found in high amount in medicinal plants, fruits, beverages and vegetables in the form of sugars conjugates mainly of glucose, rhamnose and rutinose (Herrmann, 1988) or as aglycones (Markham, 1982). A lot of research is going on in this field because of their health benefits. Flavonoids have been suggested to be called as vitamin C2 or vitamin p (Rusznyak and Szent-Györgyi, 1936). Studies revealed that they are antioxidants (Burns et al., 2000; Kaneko and Baba, 1999). In vivo and epidemiological studies showed that they are associated with reduced risk of cardiovascular diseases and certain cancers (Avila et al., 1994; Hertog et al., 1993; Khan et al., 2003; Knekt et al., 1996; Piantelli et al., 1995) (Cross et al., 1996; Gaspar et al., 1993; Qureshi et al., 2014a). Flavonoids are mainly present in the leaves and outer parts of the plants. Some times they occur in plants as aglycon, but most commonly as O-glycosides (Stobiecki, 2000) generally located at 3- or 7-position. In C- glycosides the sugar group is attached directly to the carbon atoms in ring A of the aglycon at C-6 or C-8 postion (Scalbert and Williamson, 2000; Stobiecki, 2000). More than 50 different glycosides of the most common flavonoids have been described (Hermann, 1976). The flavonoid group is sub divided into many sub groups. Flavonols, flavones, flavanols, isoflavones, flavanones and anthocyanidins are the main sub groups. They are largely planar molecules and their structural variation comes in part from the pattern of substitution: hydroxylation, methoxylation, prenylation,or glycosylation (Prasain et al., 2004). Fig. 1 shows the structure of different flavonoids under study. In flavonols, glycosylation mainly occur at 3-O and 7-O position. While 5-O glycosides are very rare for flavonoids with a carbonyl group at C-4 position; as OH group at C-5 position involve in forming a hydrogen bond with the adjacent carbonyl group at C-4 position (Prasain et al., 2004). The major flavonol aglycon found are quercetin, myricetin, kaempferol and isorhamnetin while some fruits and vegetables contain flavones such as apigenin and luteolin. There is a large number of flavonol glycosides present in plants. More than 200 glycosides of quercetin alone have been described (Harborne, 1965). Flavones also occur as glycosides but the range of different glycosides is less than in the case of the flavonols as they are lacking a 3-OH group. Glycosylation in this group mainly occurs at 7-O position. Unlike flavonols, they are also present in the form of C-glycoside in which the sugar molecule is bound to C-C bond. The presence of large number of glycosidic forms limits their quantification in plants. To overcome this problem the *Corresponding author: e-mail: mnasimuq@yahoo.com